WO2008025563A2

WO2008025563A2 - Polymer matrix, method for its production, and its use

Info

Publication number: WO2008025563A2
Application number: PCT/EP2007/007632
Authority: WO
Inventors: Ute Steinfeld; Barbara Palm; Hyeck Hee Lee
Original assignee: Kist-Europe Forschungsgesellschaft Mbh
Priority date: 2006-08-31
Filing date: 2007-08-31
Publication date: 2008-03-06
Also published as: DE102006040772A1; US20090311324A1; EP2066305A2; WO2008025563A3

Abstract

The invention relates to a polymer matrix for the specific tethering of surface epitopes or receptors of cells, where the polymer matrix has been pretreated by molecular imprinting. The invention likewise relates to a method for producing such a polymer matrix. The polymer matrices of the invention are used in therapeutic and diagnostic applications, as well as for the isolation of cells.

Description

Polymer matrix, process for their preparation and their use

The invention relates to a polymer matrix for the specific attachment of surface epitopes or

Receptors of cells, wherein the polymer matrix has been pretreated by molecular imprinting. The invention likewise relates to a process for producing such a polymer matrix. The polymer matrices according to the invention are used for therapeutic as well as diagnostic applications and also for the isolation of cells.

A targeted delivery of drugs in the body is nowadays a variety of systems that are characterized by being able to absorb much drug per particle to have a biocompatible shell and thus have low toxicity. Furthermore, the rate of delivery and effect can be adjusted. There are many different ones Varieties of particles, such as micelles, inside which the drug is trapped. The shell can be chemically modified so that early degradation in the bloodstream is avoided.

Also, liposomes, dendrimers, hydrogels and the like. Particles with the above-mentioned advantages are used for the transport of medicaments, whereby the medicament, depending on the type of particles, can be distributed both in the core and evenly distributed over the entire particle. The shell can be modified by its construction or the attachment of specific binding sites such that e.g. Cancer cells can be targeted by the particles, where they then release the drug by dissolving the particle or by chemical or physicochemical follow-up reactions. Here are pH or temperature changes called. Also, uptake of the particles into the target cells is possible, where they are then e.g. be resolved by enzymatic processes by the conditions prevailing in the cell. Due to the versatile possibilities of particle assembly, the particles can be precisely adapted to the specific requirements of the system to be treated.

A newer form of drug delivery is via Molecular Imprinted Polymers (MIP). Here, polymerization of functional and crosslinking polymers into nanoparticles takes place in the presence of the medicament to be transported. The drug serves as a template, which in a further step is washed out of the formed polymers and leaves cavities that have the specific arrangement and size for the template and thus can specifically react to it, whereby the degree of specificity can be selected. The big pros parts of MIPs are thus in the high affinity and specificity for the target molecules that correspond to those of natural receptors, their increased stability to their biological representatives as well as their ease of synthesis and adaptation to the conditions of use.

However, it is also possible to prepare the polymer in the presence of the enantiomer or of a diastereomer of the medicament (in the case of chiral active substances) or else of structural analogues. The active substance itself has thus a slightly lower affinity for the polymer, whereby the release of the active ingredient is facilitated. Another variant is conceivable in which the polymer is embossed with a substance present at the target site which has structural similarities with the active substance; The active ingredient is thus less firmly bound in the polymer and displaced by the imprinting substance at the destination. The release of the drug may be due to diffusion,

Displacement, (biological) degradation of the carrier material or an interaction of all done.

Existing MIP systems are often chosen so that they are next to the drug to be transported

Wear receptors or antibodies to recognize specific epitopes on the target cell to deliver their cargo here. They circulate through the bloodstream until they are gradually absorbed into the target cells. The Blutström serves as a direct means of transport. Such systems are known from the following publications:

0. Kotrotsiou, K. Kotti, E. Dini, 0. Kammona and C. Kiparissides; Journal of Physics: Conference Series 10 (2005) 281-284, Alexandridou S. and Kiparissides C, Proc. Of the EC-NSF Workshop on Nanotechnology-Revolutionary Opportunities and Societal Implications (Lecce, Italy, January 31-February 1, 2002),

Byrne M.E., Park K. and Peppas N.A., 2002 Adv. Drug Delivery Rev. 54, 149-61 and

Majeti N.V., Ravi Kuraar, J. Pharm. Pharmaceut. Be. 3 (2): 234-258 (2000).

Proceeding from this, it was an object of the present invention to provide an easy-to-use method with which surface epitopes or receptors of cells can be selectively recognized or bound.

This object is achieved by the polymer matrix having the features of claim 1 and the method having the features of claim 18. In claims 24 to 27 uses according to the invention are enumerated. The other dependent claims show advantageous developments.

According to the invention, a polymer matrix is provided for the specific binding of surface epitopes or receptors of cells, the polymer matrix having molecularly imprinted binding cations and epitopes or receptors specific for these surfaces.

The present invention makes it possible to use the method of molecular imprinting to produce polymer matrices, in particular polymer particles, on which receptors, antibodies or bispecific zifische antibodies are replaced. Since many tumor-associated antigens or other epitopes or receptors that are involved in diseases or may play a role in the therapy known. The method according to the invention thus replaces the previous procedure known from the prior art for embossing particle surfaces in the presence of such structures, for example antigens, epitopes or receptors, or altered or reduced segments thereof.

In accordance with the present invention, polymer matrices can be prepared which simulate bispecific antibodies by tailoring and attaching to specific epitopes of a transport cell prior to their introduction into the bloodstream. A second molecularly imprinted binding site facilitates recognition and binding to other cells, e.g. Tumor cells. This can reduce the circulation time and increase the rate of uptake at the target site if the transporting cells are chosen to respond to the affected tissue.

Preferably, the binding cavities are for immune cells, in particular T-lymphocytes, monocytes or of

Monocyte-derived macrophages and antigen-presenting cells. Likewise, these binding cavities may be for stem cells.

The polymer matrix preferably consists of a biocompatible and / or natural polymer. The biocompatible polymer is preferably selected from the group consisting of poly (meth) acrylic acid, poly (2-hydroxyethyl methacrylate), polyacrylamides, polylactides, polyglycosides, polyphosphazenes, polythioesters, polyanhydrides, poly (N-vinylpyrrolidone), Poly (hydroxyalkanoates), polyurethanes, polysiloxanes, poly (ethylene oxide), poly (vinyl alcohol), poly (ethylene), poly (methyl methacrylate), poly (ethylene glycol), polyglycolides and copolymers, blends, and mixtures thereof. The natural polymers are preferably selected from the group consisting of starch, cellulose, chitosan and copolymers, blends and mixtures thereof. It is also possible to use inorganic materials, for example silicon dioxide materials, in particular porous hollow particles thereof, titanium and alloys thereof; Aluminum, calcium, titanium and cobalt oxides, - hydroxyapatites, fluorapatites and other calcium phosphates, - aluminates, selenates and antimi- ates.

The specific binding cavities may preferably be generated by the incorporation of specific epitopes, receptors or portions thereof, and subsequently removed again from the polymer matrix. This distance may be e.g. be induced by the use of hydrolytic enzymes, by heating or appropriate chemicals. It is likewise possible to generate the specific binding cavities by incorporating specific proteins, polysaccharides, fats and / or nucleic acids into the polymer matrix and subsequently hydrolyzing them by isosomal enzymes, which makes it possible to dissolve the external structure, ie the rule of the shell, comes.

The preferred form of the polymer matrix is a particle, in which case the particle surface has the binding cavities. Such particles preferably have a particle size in the range of 10 to 1000 nm. A further preferred variant provides that the polymer matrix contains a magnetic or magnetizable material, for example iron-containing material such as magnetite, maghemite or hematite. Such particles make it possible to heat selective areas in the body to which the particles bind due to the molecularly embossed surface structuring. The heating takes place by exciting the particles with radio waves or microwaves. Depending on the extent of the induced increase in temperature, this can be used, for example, to release active substances from temperature-sensitive particles or to heat the target tissue in order to kill the corresponding cells. Such magnetic nanoparticles can either be obtained by known synthesis processes or are coated with a biocompatible and biodegradable polymer of, for example, lactic acid, urethane anhydrides, siloxane, vinyl alcohols, acrylamide, ethylene, ethyl 2-cyanoacrylate, gelatin, dextran or mixtures thereof. Another variant for the preparation provides that the magnetic material is incorporated into the polymer matrix during the polymerization.

A further preferred variant provides that the polymer matrix contains at least one active substance. This makes it possible that the effect of eg T cells at the site of action can be enhanced by release of the active ingredient. Thus, it is possible to achieve a targeted release of active substance at the site of the tumor by loading with cytostatic agents. Such drug-loaded polymer matrices, which may be in the form of particles, for example, can either be injected into the bloodstream or linked ex vivo to cells, eg T lymphocytes or monocytes, before these complexes are then injected into the bloodstream. With regard to the loading of the particles with The active ingredients are no restrictions, so both a load on the inside and on the surface is possible. In the first case, the outer embossed surface is designed to be either time-dependent or by binding to the target epitope by a changed pH in the target tissue or in certain compartments, or by exocytosis of the acidic, cytolytic content of the target. Lysosomes after binding to the cell to be treated via the T-cell receptor becomes permeable to the drug or dissolves. A classic field of application for this is cancer therapy.

The release of the active ingredient may generally be by diffusion, by (biological) degradation of the support material, and by swelling or swelling followed by diffusion or all together. The active ingredient can be released both time-controlled, by desorption, pH-dependent, by lysosomal hydrolysis and / or by magnetic interaction. In this case, a sustained release may be preferred.

On polymer matrix systems capable of changing their size by swelling or shrinking, the following environmental parameters may also cause drug release: temperature; Ionic strength; charged compounds released by the target cells that bind to functional groups in the system and cause the system to swell due to the resulting electrostatic repulsion; and electron donor compounds that can undergo charge-transfer complexes with the systems. With regard to the different release mechanisms, the different variants are briefly described again.

1) pH-dependent

Introduction of the MIP-enveloped particles into the bloodstream results in attachment to T-leukocytes and transport to the target site. The reaction of the T cell to the cancer cell leads to lysosomal release with associated pH reduction. This leads to the distribution of the drug in the pH-sensitive microgels.

When using pH-sensitive protein units, the network is split at these sites, in the other microgels there is a reversible swelling or shrinking of the network, whereby the solvent contained in the microgel is released with the drug.

2) lysosomal

For the polymer units functionalized with enzyme-sensitive sections, lysosomal hydrolysis leads to cleavage of the polymer network.

3) timed

The release takes place in simple core-shell particles by the slow dissolution of the medically loaded shell or, in the case of core-loaded particles, by the dissolution of the barrier shell. 4) pH dependent (enhanced by chain reaction)

The use of pH-sensitive particles leads to the desorption of the particle / T cell unit and subsequent re-coupling to other epitopes of the

Cancer cell to further lysosomal release. Thus, the pH in the environment is gradually lowered, which ultimately leads to the complete dissolution of the particle.

5) magnetic interaction

An alternating magnetic field leads to heating of the particles and the destruction of the polymer structure and thus to the release of the drug. In the case of the temperature-sensitive microgels, there is a contraction of the polymer and a squeezing out of the solvents contained in the interstices. Here, the temperature threshold is chosen high enough to avoid premature delivery of the drug.

The magnetic particles can be localized by magnetic detection (SQUID) in the body and allow a targeted reaction start with sufficient accumulation in the affected tissue.

An applied magnetic field can also lead to a change in the pores in the gel, an electric field to a change in the charge in the membrane and a migration of a charged drug. In both cases, a change in the swelling behavior and thus a release of the active ingredient is effected.

Furthermore, an increase in temperature and thus a release of the active substance can also be caused by ultrasound. In principle, there are no restrictions with regard to the binding of the active substance to the polymer matrix. Preferably, the interactions are based on physisorption or chemisorption with the surface of the polymer matrix. Likewise, it is also possible that the active ingredient is incorporated in the polymer matrix.

Another variant of the polymer matrix according to the invention provides that this is in the form of a hydrogel. Such hydrogels preferably have a particle size in the order of 100 to 1000 nm. For the preparation of a solution consisting of the corresponding monomer units with a crosslinker, for example tetraethylene glycol dimethacrylate (EDGMA) or pentaerythritol tetraacrylate (PETEA) and with a crosslinking agent, for example functional groups such as acrylamide units which can be attached to the amide nitrogen via aliphatic, aromatic or heteroaromatic spacers are dissolved in a mixture of water and an organic solvent such as ethanol. In the production of such hydrogels according to the invention, it is also possible by the incorporation of specific protein or polysaccharide chains, fats or nucleic acids into the polymer to form functional segments which can be hydrolyzed by lysosomal enzymes and lead to the dissolution of the microgel. Likewise, pH-sensitive protein units can be used. As free radical initiators for the preparation of the hydrogels are preferably 4, 4 'azobis (4-cyanopentanoic acid) or 2, 2' azobis (isobutyronitrile) is used. Likewise, however, a photolytic radical start by means of UV light is possible. Another preferred variant provides temperature-sensitive hydrogels, for example Poly (N-alkylacrylamides) can be prepared. Their thermosensitivity can be achieved by the variation of crosslinker used and its concentration or by the addition of co-monomers. Due to the presence of magnetite or

Maghemite during the synthesis can also produce magnetic particles that can also react to external fields. With regard to the monomers, crosslinking agents and solvents used for the polymer matrix, reference is made to claims 20 to 22.

For the production of specific cavities for the detection of certain cell types, eg T-lymphocytes, monocytes, the cells, their epitopes or regions of the epitopes are bound to a supported biopolymer surface such as gelatin or dextran. For this purpose, their previously modified antibodies can be used by binding sulfhydryl or else other functional groups to the biopolymer and the abovementioned cell types via avidin-biotin complexation by means of NeutrAvidin to the functional groups. By modifying the antibodies, eg biotinylation, it is ensured that the antibody binds to the biopolymer layer in the desired orientation. The biopolymer is bound on one side of a polymer stamp as a thin layer, thereby forming capillaries with a diameter of only a few micrometers. On the opposite side of the bound cells characteristic markers or epitopes such as the tumor marker EpCAM are fixed. The targeted arrangement of both components on the particle can thus be achieved via the spatial arrangement during the synthesis by now in the capillaries, the molecular imprinting is done. The specific cavities are formed on the standing shell in the presence of the drug-loaded particles on opposite sides. By separating the polymer halves and washing with solvents, the finished particles can be extracted.

A further preferred variant envisages that the surface epitope which produces the binding cavity in the polymer matrix is bound to a sterically demanding molecule, e.g. a polymer, or a sufficiently large particle, e.g. Gelatin particles, colloids or liposomes. This will not only form a very small number of cavities for the transport cell. The mutual obstruction of the epitope particles can also be produced by a high ionic charge of the epitope particles, which leads to repulsion. Likewise, a deliberately low loading of carrier particles, even with several cavities per polymer particle, can be achieved by a low concentration of carriers during the subsequent loading, provided that the number of cavities during the polymerisation of the polymer matrix is kept sufficiently low by the said methods.

If magnetically active colloids are used as epitope carriers, they can be brought to one side of the polymerization vessel or polymerization channel, ie when using the flow-through method, by applying a magnetic field. Upon simultaneous polymerization of the polymer matrix with the opposite bound second epitope of the target molecule, the cavities are formed on the opposite side of the target molecule accordingly. When using the above method without the presence of polymer stamps, the arrangement of the cavities is non-specific. In this case, the antibodies of the T cells are bound to gelatin particles with sizes of more than 500 nm in order to prevent imprinting of the antibody-carrying particles.

A further preferred variant provides that nanoscale embossed polymers are prepared in the first step, with short oligopeptide sections as freely movable or immobilized templates which are selected from the amino acids of the proteins of the epitope which are specific for the N- or C-terminal ends consist. These peptide-imprinted polymers, which can recognize the N- or C-terminal oligopeptides and thus the target epitope with high specificity and affinity, are then intended to serve as a template for a stable prepolymerization complex with suitable functional monomers in a second step. In a third step, these are then polymerized to more or less strongly cross-linked chains (alternatively, the functional monomers are already incorporated in chains), which are held together by the non-covalent interactions between the copolymerized functional monomers and the peptide-imprinted polymer templated and In the case of binding of the peptide-imprinted polymers to the target epitope, the polymer structure is loosened, resulting in a release of the active substance (s). This described system may also be additionally coupled with a MIP specific for immune cell epitopes. The polymer matrix according to the invention can be present in different forms depending on the production process. Which includes:

• Production of polymer monoliths and subsequent fragmentation

Grafting the imprinted polymer onto preformed particles

• Preparation of polymer beads from suspension, emulsion or dispersion polymerization

• Polymer particles bound to thin films or polymer membranes

• polymer membranes

• Surface-imprinted polymer phases: The formed complexes of the template molecules with the functional ones

Monomers bind to activated surfaces, e.g. Silicon or glass surfaces, and give after washing defined embossed structures.

With regard to the possible uses, some advantageous fields of application should be enumerated here by way of example. These include, for example, targeting, covalent or non-covalent binding to the target, and controlled release of drugs into target tissues, which is an essential aspect of modern therapies because it can dramatically reduce drug concentrations. As an area of application is here, for example, the cancer therapy in which the inhibition of receptors, e.g. epidermal growth receptor (EGFR) is an important target to suppress signal transduction. The monoclonal antibodies used hitherto have the new invention

Systems have the disadvantage of being significantly unstable. Biler are immunogenic and more expensive to produce. Another advantage of the polymer matrix according to the invention over monoclonal antibodies is the significantly cheaper production.

Another use relates to the use in diagnostics by the particles are marked with fluorescent dyes or contrast agents. The corresponding molecular imprinting of the surface allows interaction with specific target cells, e.g. in the body directly or in a blood sample.

Furthermore, a compound of the polymer matrix according to the invention for cell isolation can also be carried out. The use of particles with a magnetic core and an outer molecular-imprinted surface layer thus enables isolation of the bound cells in the magnetic field.

The subject according to the invention is intended to be explained in more detail with reference to the following example, without wishing to restrict it to the specific embodiment shown here.

example 1

Analogous to the specification of Chen et al. (Jian-Feng Chen, Hao-Min Ding, Jie-Xin Wang, Lei Shao, Biomaterials 25 (2004) 723-727), hollow silica nanoparticles are prepared as carrier material for the selected active ingredient and its controlled release However, the sodium silicate component (Na ₂ SiO ₃ .9 H ₂ O) over a longer period (5-10 days) by means of a peristaltic pump to Calciumcarbo- natsuspension (particle size 90 nm) was added dropwise. In The hollow silica nanoparticles thus obtained can now be prepared according to the instructions of Seilegren et al. (Bärbel Rücker, Andrew J. Hall and Börje Sellergren, J. Mater. Chem., 2002, 12, 2275-2280) introduced p- (chloromethyl) phenyl groups and coupled them to the diethyldithiocarbamate groups as radical initiators. The carbamate-derivatized sites are now used as starting points for the UV-controlled grafting of molecularly imprinted poly (methacrylic acid-co-ethylene glycol dimethacrylate) copolymers, serving as templates

Oligopeptides contained in the N- or C-terminal sequences of the respective target epitopes and specific for the selected epitopes.

Claims

claims

1. Polymer matrix for the specific binding of surface epitopes or receptors of cells, the polymer matrix having molecularly imprinted and for these surface epitopes or receptors specific binding cavities.

2. Polymer matrix according to claim 1, characterized in that the polymer matrix have molecularly imprinted binding cavities for at least one immune cell, in particular T lymphocytes, monocytes or monocyte-derived macrophages, antigen-presenting cells or for at least one stem cell.

3. Polymer matrix according to the preceding claim, characterized in that the polymer matrix is formed from a biocompatible and / or natural polymer.

4. Polymer matrix according to the preceding claim, characterized in that the biocompatible

Polymers are selected from the group consisting of poly (meth) acrylic acid, poly (2-hydroxyethyl methacrylate), polyacrylamides, polylactides, polyglycosides, polyphosphazenes, polythioesters, polyanhydrides, poly (N-vinylpyrrolidone), poly (hydroxyalkanoates), polyurethanes, polysiloxanes, poly (ethylene oxide), poly (vinyl alcohol), poly (ethylene), poly (methyl methacrylate), poly (ethylene glycol), polyglycolides and Copolymers, blends and mixtures thereof and the natural polymers are selected from the group consisting of starch, cellulose, chitosan and copolymers, blends and mixtures thereof.

5. Polymer matrix according to one of the preceding claims, characterized in that the specific binding cavities have been generated by the incorporation of specific epitopes, receptors or parts thereof and subsequently removed again from the polymer matrix, e.g. through the use of hydrolytic enzymes, heat or appropriate chemicals.

6. Polymer matrix according to one of the preceding claims, characterized in that the specific binding cavities were produced by incorporation of specific proteins, polysaccharides, fats and / or nucleic acids with subsequent hydrolysis by lysosomal enzymes.

7. Polymer matrix according to one of the preceding claims, characterized in that the polymer matrix contains a magnetic or magnetisable material, in particular iron-containing material such as magnetite, maghemite or hematite.

8. Polymer matrix according to one of the preceding claims, characterized in that the polymer matrix contains at least one active ingredient.

9. Polymer matrix according to the preceding claim, characterized in that the active ingredient is time-controlled, temperature-controlled, by desorption, controlled by the ionic strength, pH-dependent, initiated by lysosomal hydrolysis, by compounds released by the cells, initiated by Elektrodonorverbindungen and / or releasable by magnetic interaction.

10. Polymer matrix according to one of claims 8 or 9, characterized in that the active substance is retargeted releasable.

11. Polymer matrix according to any one of claims 8 to 10, characterized in that the at least one active substance by physical or chemisorption is superficially bound to the polymer matrix and / or incorporated into the polymer matrix.

12. Polymer matrix according to one of the preceding claims, characterized in that the polymer matrix contains at least one contrast agent and / or at least one dye.

13. Polymer matrix according to one of the preceding claims, characterized in that the polymer matrix is present in the form of a particle and the particle keloberflache has the binding cavities.

14. Polymer matrix according to claim 13, characterized in that the particles have a particle size in the range of 10 to 1000 nm.

15. Polymer matrix according to one of the preceding claims, characterized in that the polymer matrix is in the form of a hydrogel.

16. Polymer matrix according to the preceding claim, characterized in that the active substance release is effected by the pH sensitivity of the hydrogel.

17. Polymer matrix according to claim 15, characterized in that the active substance release by the temperature sensitivity of the

Hydrogel takes place.

18. A process for the preparation of a polymer matrix having binding cavities for surface epitopes or receptors of cells, in which a polymer matrix is produced by cross-linking and surface-bound by physical and / or chemisorption specific proteins, polysaccharides, fats and / or nucleic acids to the matrix and / / or incorporated into the matrix.

19. The method according to claim 18, characterized in that the polymer matrix is prepared from at least one monomer and at least one crosslinker in the presence of a radical initiator in a solvent.

20. The method according to claim 19, characterized in that the polymer matrix of monomers selected from the group consisting of carboxylic acids (in particular (meth) acrylic acid, trifluoromethacrylic acid, vinyl benzoic acid, itaconic acid) and their amides, sulfonic acids (especially acrylamidomethylpropanesulfonic acid), heteroaromatic Bases (in particular vinylpyridines, vinylimidazoles, vinylpyridines, vinylpurines), aliphatic and aromatic vinyl compounds with chelating groups (in particular iminoacetic acid, ethylenediaminetetraacetic acid) and mixtures of these

Monomers is produced.

21. The method according to any one of claims 19 or 20, characterized in that as cross-linker compounds selected from the group consisting of divinylbenzene crosslinker, (meth) acrylic acid crosslinkers, tri- and tetra-functional crosslinkers, acrylamide crosslinkers and mixtures thereof.

22. The method according to any one of claims 19 to 21, characterized in that as a solvent in the polymerization aliphatic or alicyclo metallic hydrocarbons, in particular hexane, heptane or cyclohexane, aromatic hydrocarbons hydrogens, in particular toluene, halogenated hydrocarbons, in particular chloroform, dichloromethane or 1, 2-dichloroethane, alcohols, in particular, methanol, ethanol or propanol, ethers, acetonitrile, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, dioxane, dimethyl sulfoxide and theirs Mixtures with each other and with water is used.

23. The method according to any one of claims 19 to 22, characterized in that as initiator for the polymerization of radical initiator, in particular 2, 2'-azobis-isobutyronitrile or 2,2'-azobis (2, 4, dimethylvaleronitrile) and / or UV Radiation is used.

24. Use of the polymer matrix according to any one of claims 1 to 17 for the inhibition of receptors for therapeutic purposes.

25. Use of the polymer matrix according to one of claims 1 to 17 for target-oriented transport and the release of active ingredients.

26. Use of the polymer matrix according to claim 12 for the diagnosis of e.g. Pathogens.

27. Use of the polymer matrix according to claim 7 for the isolation of cells in the magnetic field.